Pakladok T et al. (2014), Up-regulation of hERG K⁺ channels by B-RAF.

XB-ART-48483

PLoS One 2014 Jan 01;91:e87457. doi: 10.1371/journal.pone.0087457.

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Up-regulation of hERG K⁺ channels by B-RAF.

Pakladok T , Hosseinzadeh Z , Almilaji A , Lebedeva A , Shumilina E , Alesutan I , Lang F .

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Human ether-a-go-go related-gene K⁺ channels (hERG) participate in the regulation of tumor cell proliferation and apoptosis. HERG channel activity is up-regulated by growth factors. Kinases sensitive to growth factor signaling include the serine/threonine protein kinase B-RAF. The present study thus explored whether B-RAF influences hERG channel expression and activity. To this end, hERG channels were expressed in Xenopus oocytes with or without wild-type B-RAF, hERG channel activity was determined utilizing dual-electrode voltage clamp and hERG protein abundance in the cell membrane was analyzed utilizing confocal microscopy as well as chemiluminescence. Moreover, in rhabdomyosarcoma RD cells the effect of B-RAF inhibitor PLX-4720 on hERG-mediated current was quantified by whole-cell patch clamp and hERG cell surface protein abundance by utilizing biotinylation of cell surface proteins as well as flow cytometry. As a result, co-expression of wild-type B-RAF in hERG-expressing Xenopus oocytes significantly increased hERG channel activity and hERG channel protein abundance in the cell membrane. Treatment for 24 hours of B-RAF and hERG-expressing Xenopus oocytes with B-RAF inhibitor PLX-4720 (10 µM) significantly decreased hERG-mediated current and hERG cell surface expression. Similarly, in rhabdomyosarcoma RD cells, treatment for 24 hours with B-RAF inhibitor PLX-4720 significantly decreased hERG cell membrane protein abundance and hERG-mediated current. In conclusion, B-RAF is a powerful regulator of hERG channel activity and cell surface hERG protein abundance.

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Species referenced: Xenopus
Genes referenced: akt1 braf gnao1 kcnh1 kcnh2
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	Figure 1. Coexpression of B-RAF increased hERG current in Xenopus oocytes. A. Original tracings recorded in Xenopus oocytes injected with water (a), with cRNA encoding hERG alone (b) or with cRNA encoding hERG together with wild-type B-RAF (c). The Xenopus oocytes were depolarized from −80 mV holding potential to different voltages followed by a 500 ms repolarization to −60 mV evoking outward tail currents. B. Arithmetic means ï¿½ SEM (n = 12ï¿½47, arbitrary units) of the normalized outward tail current following a depolarization to +70 mV, recorded in Xenopus oocytes injected with water (white bar), with cRNA encoding hERG alone (light grey bar), or with cRNA encoding both, hERG and wild-type B-RAF (black bar). ***(p<0.001) indicates statistically significant difference from Xenopus oocytes expressing hERG channels alone. C. Arithmetic means ï¿½ SEM (n = 12ï¿½47, nA) of the peak tail current as a function of voltage in Xenopus oocytes injected with water (black triangles), with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG and wild-type B-RAF (black circles). D. Arithmetic means ï¿½ SEM (n = 22ï¿½47, arbitrary units) of the normalized peak tail current as a function of voltage in Xenopus oocytes injected with cRNA encoding hERG alone (white circles) or with cRNA encoding hERG together with wild-type B-RAF (black circles).
	Figure 2. Coexpression of B-RAF increased hERG-HA protein abundance at the surface of hERG-expressing Xenopus oocytes. A. Confocal images of hERG-HA protein cell surface expression in Xenopus oocytes injected with water (left panel), with cRNA encoding hERG-HA alone (middle panel) or with cRNA encoding hERG-HA together with wild-type B-RAF (right panel). Images are representative of three independent experiments. B. Arithmetic means ï¿½ SEM (n = 81ï¿½93, arbitrary units) of hERG-HA protein abundance in the cell membrane measured by chemiluminescence in Xenopus oocytes injected with water (white bar), with cRNA encoding hERG-HA alone (light grey bar), or cRNA encoding hERG-HA and wild-type B-RAF (black bar). ***(p<0.001) indicates statistically significant difference from Xenopus oocytes expressing hERG channels alone.
	Figure 3. B-RAF inhibitor PLX-4720 decreased hERG current in Xenopus oocytes co-expressing hERG and B-RAF. A. Original tracings recorded in Xenopus oocytes injected with water (a), with cRNA encoding hERG alone (b) or with cRNA encoding hERG together with wild-type B-RAF without (c) and with (d) treatment with B-RAF inhibitor PLX-4720 (10 ï¿½M, 24 hours). The Xenopus oocytes were depolarized from −80 mV holding potential to different voltages followed by a 500 ms repolarization to −60 mV evoking outward tail currents. B. Arithmetic means ï¿½ SEM (n = 12ï¿½46, arbitrary units) of the normalized outward tail current following a depolarization to +70 mV, recorded in Xenopus oocytes injected with water (white bar), with cRNA encoding hERG alone (light grey bar), or with cRNA encoding hERG together with wild-type B-RAF without (black bar) and with (dark grey bar) treatment with B-RAF inhibitor PLX-4720 (10 ï¿½M, 24 hours). ***(p<0.001) indicates statistically significant difference from Xenopus oocytes expressing hERG channels alone; ###(p<0.001) indicates statistically significant difference from Xenopus oocytes expressing hERG together with B-RAF without treatment with PLX-4720. C. Confocal images of hERG-HA protein cell surface expression in Xenopus oocytes injected with water (first panel), with cRNA encoding hERG-HA alone (second panel) or with cRNA encoding hERG-HA together with wild-type B-RAF without (third panel) or with (last panel) treatment with B-RAF inhibitor PLX-4720 (10 ï¿½M, 24 hours). Images are representative of three independent experiments.
	Figure 4. B-RAF inhibitor PLX-4720 decreased hERG protein abundance at the cell surface in rhabdomyosarcoma RD cells. A. Representative original western blot showing hERG membrane protein abundance (anti-Kv11.1 antibody, Alamone Labs) analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (PLX-4720). B. Arithmetic means ï¿½ SEM (n = 7, arbitrary units) of normalized hERG membrane protein abundance analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (black bar). (p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone. C. Representative original dot plots of hERG-FITC positive cells at the cell surface analysed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (PLX-4720); FL-1 Height: hERG-FITC fluorescence intensity. D. Arithmetic means ï¿½ SEM (n = 5, %) of normalized percentage of positive cells showing hERG expression at the cell surface analyzed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (black bar). (p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.
	Figure 5. B-RAF inhibitor PLX4720 decreased hERG currents in rhabdomyosarcoma RD cells. A. Inward currents elicited in a bath solution containing 40: the membrane potential was held at −80 mV and then after the preconditioning step from −80 mV to +60 mV for 2 s stepped to the test potential of −120 mV for 500 ms. The currents were measured in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (a) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (b). B. Mean peak current density ï¿½ SEM (n = 5ï¿½12) plotted against the precondition potential in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white cycles) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (black cycles). C. Mean peak current density ï¿½ SEM (n = 5ï¿½12) measured at −120 mV after the precondition potential to +50 mV in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 ï¿½M B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.

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Alesutan, Upregulation of Na-coupled glucose transporter SGLT1 by Tau tubulin kinase 2. 2012, Pubmed, Xenbase